This proposal will continue to investigate the Th1 and Th2 mediated responses that regulate the induction and progression of proteoglycan-induced arthritis (PGIA). Development of PGIA correlates with an increase in IFN-gamma production suggesting that it is a Th1-mediated disease. The production of IFN-gamma directly impacts disease severity based on our findings that disease is suppressed when IFN-gamma is either neutralized or absent (IFN-gamma+ mice). We assessed the IL-12/Stat4 activation pathway and demonstrated that disruption of Stat4 gene (Stat4 /) increased resistance to PGIA. Interestingly, Stat4-/- mice were even more resistant to PGIA than IFN-gamma-/- mice. We reasoned that factors regulated by Stat4, other than IFN-gamma, must contribute to susceptibility to PGIA. In addition, since IFN-gamma is produced in Stat4-/- mice there must be a Stat4-independent mechanism of IFN-gamma production in PGIA. The earliest signal for activation of Th1 responses is hypothesized to be the transcription factor T-bet (T-box expressed in T cells). We found that T-bet is expressed rapidly after PG immunization in Wt but not in IFN-gamma-/- or Stat4-/- mice. Thus, we are interested in understanding if T-bet is the initiator of Th1 responses that control susceptibility to PGIA. In addition to characterizing the Th1 immune responses, we have identified several mechanisms by which IL-4 controls PGIA. Enhanced disease severity in IL-4-/- mice was associated with an increase in IL-12, TNF-alpha and IFN-gamma production. In addition to the rise in cytokines associated with type 1 responses, the PG-specific IgG2a antibody levels were considerably increased. Our studies have broadly examined the regulation of PGIA by IL-4 and IFN-gamma but since IL-4 and IFN-gamma receptors are expressed on several different cell populations (macrophages, T cells and B cells), it is possible that a specific cell population is the primary target in controlling disease severity. Therefore, it seems of utmost importance to identify the contribution of different cell types bearing either the IFN-gammaR or IL-4R to the inflammatory process. To address these issues, in this proposal we will (i) characterize the relationship between IFN-gamma and Stat4- regulated molecules in PGIA and determine if the Stat4-regulated molecules, CCR5 and PSGL-1, contribute to PGIA, (ii) determine the role of IL-23 and IL-27 in the Stat4-independent activation of IFN-gamma expression, (iii) determine whether the transcription factor T-bet critically regulates the initial induction of Th1 responses in arthritis, and (iv) identify the cell population (macrophages, T cells, or B cells) targeted by IL-4 and IFN-gamma that regulates inflamrhation utilizing deletion of either IL-4Ralpha or IFN-gammaR in a cell-specific manner by Cre-mediated recombination. Our overall hypothesis is that multiple proteins regulate PGIA; novel and previously identified (cytokines, chemokines, adhesion molecules transcription factors) that synergistically control arthritis onset and severity, and thus, provides attractive targets for the development of novel therapeutics.